|
Generation and analysis of double deficient transgenic mice for kallikrein-related peptidase 5 and kallikrein-related peptidase 14
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Fulková, Helena (referee)
Kallikrein-related peptidases (KLKs) constitute a highly conserved serine protease family. Based on in vitro experiments, KLKs are predicted to play an important role in a number of physiolog- ical and pathophysiological processes. However, their role in vivo remains not fully understood, partially due to a lack of suitable animal models. In this work, we aim to prepare a KLK5 and KLK14 double-deficient mouse model. Both KLK5 and KLK14 were proposed to be involved in epidermal proteolytic networks critical for maintaining skin homeostasis. However, both KLK5 and KLK14 single-deficient mouse models show minimal or no phenotype, likely due to similar substrate specificity resulting in functional compensation. Double-deficient mice cannot be easily obtained by crossing due to localization of the Klk5 and Klk14 genes within the same locus on chromosome 7. We report that KLK5 and KLK14 double-deficient mice were success- fully generated, mediated by transcription activator-like effector nucleases (TALENs) targeting Klk14 by microinjection of TALEN mRNA into KLK5-deficient zygotes. Furthermore, we show that KLK5 and KLK14 double-deficient mice are viable and fertile. We believe that these novel mouse models may serve as a useful experimental tool to study KLK5 and KLK14 in vivo.
|
|
Functional analysis of posttranscriptional gene regulation by TENT5A in biomineralization and metabolism
Aranaz Novaliches, Goretti ; Sedláček, Radislav (advisor) ; Hovořáková, Mária (referee) ; Tencerová, Michaela (referee)
Non-canonical poly-A polymerases, such as TENT5A, belong to the Terminal nucleotidyl transferases (TENTs) family and are crucial for mRNA protection, stability, and translation. A Tent5a knock-out (KO) mouse model was generated in our laboratory, which exhibited a phenotype in teeth, skeleton structure, and metabolism. In my PhD project, I aimed to characterize the molecular mechanism underlying these phenotypes and explore their potential connection to rare human diseases. I focused on the biological function of Tent5a gene in enamel development (amelogenesis) and mRNA stabilization. Micro-computed tomography and scanning electron microscopy revealed that Tent5a KO mice displayed thin, hypomineralized enamel with disrupted microstructure, a condition known as Amelogenesis imperfecta. Direct mRNA sequencing demonstrated that TENT5A is responsible for polyadenylation of amelogenin (AmelX) and other secreted proteins, leading to a shortened poly-A tail in Tent5a KO ameloblasts. Moreover, Tent5a KO mice disclosed impaired self- assembly of enamel matrix proteins (EMPs) such as AMELX and ameloblastin (AMBN), leading to compromised hydroxyapatite deposition and enamel formation. In addition to its role in teeth, I investigated the physiological functions of EMPs in other tissues, considering that EMP...
|
|
4th CCP Phenogenomics Conference abstract book
Sedláček, Radislav
The fourth CCP Phenogenomics Conference was held as a hybrid meeting. The scientific committee selected the topic of rare diseases: experimental models & delivery of therapies as the main thematic focus of the 2022 Conference. The Conference provided again an excellent opportunity to support networking and interactions among the researchers, CCP staff, users and experts from the commercial sector.
|
|
Mouse models for Angelman syndrome: generation and characterization
Syding, Linn Amanda ; Sedláček, Radislav (advisor) ; Vyklický, Ladislav (referee) ; Valeš, Karel (referee)
Angelman syndrome (AS) is a neurodevelopmental disease found in 1 to 10,000 to 40,000 births, exhibiting an equal gender ratio. Key characteristics of the disease include an ataxic gait with tremor, severe mental retardation, profound speech impairment and seizures. Behavioral deficits such as increased anxiety and autism spectrum disorder features is found in affected individuals as well. The disease stems from the imprinted region 15q11.2-13q where genes are either maternally or paternally expressed as a result of parent-of-origin specific expression of the alleles. There are four main genetic etiologies causing AS namely, i) a large deletion ranging from 4-6 Mb on the maternally inherited allele including imprinted and bi-allelically expressed genes, ii) maternal deletion of the Ubitiquin ligase E3 (UBE3A) gene, iii) paternal uniparental disomy and iv) imprinting defect leading to inappropriate methylation of the locus. So far, there is no cure for AS rather the symptoms are ameliorated using a multidisciplinary approach. The goal of the doctoral study was to further decipher the role of Ube3a and Gabra5 using two mouse models to gain more knowledge about the involvement of these two genes for future therapeutic interventions in for Angelman syndrome. One model generated was a full gene deletion...
|
|
4th CCP Phenogenomics Conference abstract book
Sedláček, Radislav
The fourth CCP Phenogenomics Conference was held as a hybrid meeting. The scientific committee selected the topic of rare diseases: experimental models & delivery of therapies as the main thematic focus of the 2022 Conference. The Conference provided again an excellent opportunity to support networking and interactions among the researchers, CCP staff, users and experts from the commercial sector.
|
|
3rd CCP Phenogenomics Conference 2021: Abstract Book
Sedláček, Radislav
The Conference was divided into two blocks. The first day was devoted to the theme “Human diseases and models“. The second day was specifically dedicated to preclinical development, including covid-19, which focused on translation of the basic research into the application. The Conference provided an excellent opportunity to support networking and interactions among the CCP users and experts.
|
|
2nd CCP Phenogenomics Conference 2020: Abstract Book
Sedláček, Radislav
The Conference was divided into two blocks. The first day was devoted to the theme “Human diseases and models“. The second day was specifically dedicated to preclinical development, including covid-19, which focused on translation of the basic research into the application. The Conference provided an excellent opportunity to support networking and interactions among the CCP users and experts.
Plný tet: PDF
|
|
Genome editing using programmable endonucleases
Hanečková, Radmila ; Sedláček, Radislav (advisor) ; Sýkora, Michal (referee)
Programmable endonucleases are engineered proteins that recognize specific nucleotide sequences and that are capable of introducing double-strand breaks within these sequences. Zinc-finger nucleases have been used extensively as a tool in genome editing, the practice of introducing changes into genomes of cell lines or whole organisms as a way to study gene function. Recently, new types of programmable endonucleases have emerged in the form of transcription activator like effector (TALE) nucleases and the CRISPR/Cas system. The types differ in respect to their mechanism of function, accessibility, selectivity, frequency of off-target cleavage and cytotoxic effects. Here, we compare zinc-finger nucleases, TALENs and the CRISPR/Cas system and explore their current and possible future applications in a broad spectrum of research ranging from developing genetically modified organisms to gene therapy. Powered by TCPDF (www.tcpdf.org)
|
| |
| |